In this study, we have analyzed the cold shock-induced changes in the OM proteome of M. catarrhalis and identified TbpB, whose expression was increased more than two-fold after a 26°C cold shock, as a member of the iron acquisition systems that is important for both growth and virulence. Our data demonstrate that the expression of transferrin receptors and transferrin binding on the bacterial surface were also increased when M. catarrhalis was exposed to a 26°C cold shock. Transferrin is predominantly found in serum and in serous exudates. During pronounced inflammation, it is likely that the local tissue damage results in the transsudation of various iron sources, including transferrin, to mucosal surfaces acting as additional iron sources for M. catarrhalis .
We also demonstrate that a 26°C cold shock increased the expression of genes involved in lactoferrin acquisition and enhanced lactoferrin binding on the surface of M. catarrhalis. The overall presence of lactoferrin receptors in M. catarrhalis isolates suggests its important role in colonization or infection . In our previous study we demonstrated that exposure of M. catarrhalis to 26°C increases the release of proinflammatory cytokine IL-8 in pharyngeal epithelial cells likely leading to the increased inflammation . Thus, greater local concentrations of IL-8 would promote enhanced recruitment and influx of neutrophils that release lactoferrin from their secondary granules, which contribute to lactoferrin levels both locally and in the circulation [33, 34]. On the other hand, increased expression of M. catarrhalis lactoferrin binding proteins following cold shock would facilitate the binding and acquisition of iron from lactoferrin to support growth of bacteria in the mucosal environment. It has been shown that supplemental lactoferrin can enhance the virulence of meningococcal infection in mice . In addition to iron acquisition, lactoferrin receptors may provide protection against anti-bacterial cationic peptides (eg, lactoferricin) and reduce complement-mediated killing. The pneumococcal surface protein PspA binds lactoferrin and protects Streptococcus pneumoniae against the antibacterial effect of lactoferricin . The release of LbpB from the cell surface by a NalP protease protects Neisseria meningitidis against bactericidal antibodies . Therefore, increased expression of lactoferrin receptors and enhanced binding of lactoferrin on the surface of bacteria following cold shock might be associated with enhanced protection of M. catarrhalis against anti-bacterial cationic peptides and bactericidal antibodies.
The level of UspA2 protein that afforded serum resistance in the bactericidal activity assay has been shown to correlate with increased binding of vitronectin . Our results indicate that cold shock upregulates the UspA2 protein expression and promotes M. catarrhalis binding to vitronectin. Increased UspA2 protein expression at 26°C was not the result of higher copy number of uspA2 mRNA, indicating that post-transciptional mechanisms are involved in upregulation of this protein after cold shock . Cold shock did not influence the serum resistance of O35E strain indicating that M. catarrhalis strains may need to maintain a certain threshold level of UspA2 protein necessary to evade host defenses. Most seroresistant M. catarrhalis strains express at 37°C sufficient levels of UspA2 to mediate serum resistance . It is conceivable that cold shock would increase UspA2 expression and vitronectin binding in M. catarrhalis strains constitutively expressing low levels of UspA2, leading to the enhanced serum resistance.
The infant population during the first year of life possesses a substantial proportion of IgD in saliva . The interaction between Hag and IgD mediates B cell endocytosis and killing of M. catarrhalis whereas non-IgD-binding bacteria were not taken up by B cells . Furthermore, IgD-stimulated mucosal basophils release antimicrobial factors inhibiting the replication of M. catarrhalis . Here we demonstrate that cold shock at 26°C reduces the mRNA expression level of hag, Hag protein expression and the Hag-mediated binding of human IgD to the surface of M. catarrhalis. Decreased copy numbers of hag at 26°C were also found in other clinical isolates indicating that this effect is a general characteristic of seroresistant M. catarrhalis . Therefore, reduced expression of Hag and decreased binding of IgD on the bacterial surface following cold shock might lead to reduced stimulation of B cells and increased survival by prevention of endocytosis by these cells as well as to decreased stimulation of basophils leading to reduced release of antimicrobal factors. However, the presence of specific IgD against LOS triggered increased recognition of bacteria following cold shock (Figure 6). Consequently, children who lack LOS-specific IgD may be more susceptible to M. catarrhalis infections, particularly after exposure to cold air.
Three OMPs were found to be differentially (a greater than two fold change) regulated in response to a 26°C cold shock (Figure 1), while immunoblot and flow cytometric analysis revealed that several other OMPs are also involved in cold shock response. The lack of some differentially regulated OMPs in the 2-DE pattern might be the result of difficult identification or low abundance. Furthermore, protein spots with a fold change below the indicated threshold were considered by the Image Master 2D program as not relevant.
Thus, cold shock, which occurs when humans breathe cold air , is a physiologic phenomenon during the cold season and entails a range of adaptive events in the residential upper respiratory tract flora that lead to the stimulation of nutrient (e.g., iron)-acquistion, serum resistance and immune evasion potentially resulting in increased bacterial density on the nasopharyngeal surface. Clinical studies in children have demonstrated that the density of M. catarrhalis in the nasopharynx is positively associated with prolonged respiratory tract symptoms and a greater likelihood of purulent otitis media [40, 41]. This study demonstrates that a 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates both CopB and UspA2 expression, the latter leading to improved vitronectin binding on the surface of bacteria. In contrast, cold shock decreases the expression of Hag and reduces the IgD-binding on the surface of M. catarrhalis. These findings indicate that cold air in the human upper respiratory tract induces in M. catarrhalis a complex of adaptive mechanisms that may enable the bacterium to target their host cellular receptors or soluble effectors and consequently to display enhanced growth, colonization and virulence.